Pongsada Prasonguppatum, MD¹, Sumolrat Panthong, MSc¹, Chunya Udomwong, MD¹

Background: High volumes of formalin-fixed paraffin-embedded (FFPE) samples are processed for Mycobacterium tuberculosis (MTB) real-time polymerase chain reaction (RT-PCR) testing, making manual DNA extraction prone to human error and cross-contamination. Automated nucleic acid extraction offers a more efficient alternative.

Objective: To compare false positive rates, invalid results, DNA yield, and purity between automated and manual DNA extraction methods for MTB RT-PCR.

Materials and Methods: One thousand three hundred eighteen FFPE samples were evaluated by a pathologist for tissue reactions and classified into histologic scores. Scores of 0, 1, 2, and 3 indicated no reaction, non-specific inflammation, non-necrotizing granuloma or caseous necrosis without granuloma, and necrotizing granuloma, respectively. Of these, 767 (58.19%) underwent manual extraction, and 551 (41.81%) underwent automated extraction. RT-PCR was performed to detect MTB, with false positives identified by reviewing PCR-positive samples that did not align with the histological scores. False positivity due to cross-contamination was confirmed if a repeat PCR test, performed on newly extracted DNA, yielded a negative result. DNA yield and purity were compared between the methods using a Mann-Whitney U test.

Results: False positive rates were 1.69% for manual extraction and 0.91% for automated extraction, with invalid result rates of 2.09% and 3.27%, respectively. The manual method yielded higher median (IQR) DNA concentration and yield than the automated method at 334.60 (113.00 to 862.20) versus 120.80 (30.40 to 382.60) ng/μL and 10,038.00 (3,390.00 to 25,866.00) versus 6,040.00 (1,520.00 to 19,130.00) ng, respectively. DNA purity was also higher with the manual method with A260/A230: 2.22 (2.12 to 2.27) versus 2.02 (1.39 to 2.23), A260/A280: 1.94 (1.90 to 1.97) versus 1.90 (1.85 to 1.93). All differences were statistically significant (p<0.0001).

Conclusion: Automated nucleic acid extraction reduced false positive rates by 0.78% but increased invalid result rates by 1.18%. It yielded lower DNA yield and purity compared to manual extraction. Despite these limitations, automation remains a practical option for high-throughput processing, offering substantial time and resource savings with manageable invalid result rates.

Received 22 September 2025 | Revised 6 December 2025 | Accepted 12 December 2025

DOI: 10.35755/jmedassocthai.2026.1.03467